PROJECT SUMMARY (DESCRIPTION) Heme and the regulation of its biosynthesis are important for normal cell function, and contribute to the success of bacteria to diverse environments, including those associated with pathogenic and symbiotic interactions with higher eukaryotes. Heme is now known to be a regulatory molecule that allows cells to sense and adapt to environmental cues to maintain homeostasis. The broad objective of the proposed work is to understand how heme mediates the control of gene expression, with an emphasis on iron metabolism, and to reconcile this role for heme with its toxicity in cells. The heme biosynthetic pathway culminates with the insertion of iron into a protoporphyrin ring by a reaction catalyzed by ferrochelatase. We discovered a regulatory protein called Irr (iron response regulator) from the bacterium Bradyrhizobium japonicum that interacts directly with ferrochelatase. This novel mechanism allows Irr to respond to heme locally at the site of heme synthesis. Irr is a conditionally stable protein that functions under iron limitation, but degrades in response to iron in a heme-dependent manner. Irr was initially described as a regulator of heme biosynthesis, but new evidence reveals that it is a global regulator of iron-dependent genes. Thus, a major objective of this proposal is to address the hypothesis that B. japonicum senses iron through the status of heme in an Irr-dependent manner to regulate iron homeostasis and metabolism. Furthermore, Irr homologs are found in most -Proteobacteria, and thus the proposed work serves as a model for understanding heme and iron metabolism in pathogenic bacteria that are experimentally much less tractable than B. japonicum. Three specific aims are proposed. 1. Determine the mechanism by which Irr recognizes target genes and controls their expression. Irr strongly regulates genes encoding ferric iron transporters and many other iron- related genes, and is both a positive and negative regulator. We are particularly interested in activation by Irr since positive control of bacterial iron transport is uncommon. 2. Elucidate the mechanism by which Irr senses the status of heme. Irr interacts with ferrochelatase, which provides the regulatory input to Irr. We will characterize this interaction both biochemically and genetically, and elucidate the subsequent inhibition and degradation of Irr. 3. Elucidate the regulation of heme utilization genes by iron by an Irr-independent mechanism. Data suggest that heme synthesized de novo is controlled differently than that acquired exogenously. We will identify this regulatory mechanism, and establish how Irr-dependent and -independent metabolism is integrated.